Electrofusionof cells and apparatus therefore

ABSTRACT

The present invention relates to a method and apparatus for fusing first and second cells. In particular, the method includes positioning the first and second cells between two electrodes ( 35 ) in a fluid filled container ( 40 ) using a pipette system ( 33 ), with the first and second cells being held separated from each electrode. Once this has been achieved a current having a predetermined waveform is applied to the electrodes ( 35 ) to generate a predetermined electric field thereby causing the cells to fuse.

BACKGROUND OF THE INVENTION

The present invention relates to a method and apparatus for fusing firstand second cells, and in particular, for producing hybrid cells byelectrofusion.

DESCRIPTION OF THE PRIOR ART

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgement or any form of suggestion that theprior art forms part of the common general knowledge in Australia.

Previously it has be known to fuse cells together using a variety oftechniques such as chemical fusion employing polyethylene glycol,biological methods such as viruses or viral proteins or electrofusion ofcells in suspension. Methods of fusion carried out by chemical orbiological means often suffer from problems associated withcontamination, low efficiency and cytotoxity.

There are a number of advantages to using electrofusion for producinghybrid cells. The fusion conditions can be better controlled andoptimised depending on the type of cell to be fused than chemical orbiological fusion allows. This allows electrofusion to lead to anincrease in cell fusion efficiency.

The basis for electrofusion is to expose pairs of cells, in closemembrane contact, to an electric field that induces a sufficient voltageacross their cellular membrane to cause mechanical breakdown of the cellmembrane and the formation of pores at the point of cell-to-cellcontact. Ideally the pores should be of sufficient size to allowtransfer of cellular material between the two cells, particularlyallowing the two nuclei to come together and subsequently fuse. Thisformation of pores should also be reversible such that any pores formedat points other than that of cell-to-cell contact seal quickly.

A further process known as dielectropherisis (DEP) in which anon-uniform alternating electric field is applied to the cells in orderto ensure good cellular contact prior to fusion. Typically DEP isemployed to form ‘pearl chains’ of large numbers of cells between theelectrodes in the fusion container.

Whilst electrofusion has a number of advantages over chemical andbiological processes, all of these methods fuse cells simultaneously inlarge numbers. The pairing of the cells, necessary to form hybrid cells,is therefore completely random. Accordingly, the current methods arelimited in that they require a chemically sensitive immortal cell suchthat unfused and self-fused cells can be eliminated from the finalculture.

Furthermore, these ‘bulk’ methods also do not lend themselves to hybridcreation from rare cells. Typically fusion methods require millions ofcells in order to overcome the problems associated with ensuringcellular contact between the desired cells. In some cases the number oftarget cells for fusion might only number in 10's-100's. Further, in therecovery phase, whereby cells from the fusion process are plated out andthe chemical selection process to remove unfused cells takes place,there is no guarantee of clonal purity in the final product. Thisplating of cells is also extremely time consuming.

It would therefore be desirable to provide a method whereby lessernumbers of cells could be selected, electrically fused and recovered togrow.

An example of a system suitable for performing electrofusion on a smallnumber of cells is described in WO93/05166. This describes apparatusthat utilises an electrode coated with ligands. In use, the ligands areused to attract target cells bearing complimentary ligands. Once theligands are bound, the cells are therefore effectively bound to theelectrode. Accordingly, at this point the target cells can be broughtinto contact with partner cells allowing the cells to be fused.

However a number of drawbacks exist with these techniques. Firstly, thetarget cell is held in contact with the electrode during theelectrofusion process. As a result the cell is usually subject to anintense electric field which tends to damage the cell. Secondly, thetechnique can only be performed with a number of target cells attachedto the electrodes, and a number of partner cells. Accordingly, thismeans that any cells successfully fused may be separated out from cellsthat do not fuse, which can be a complex procedure. A furtherdisadvantage of this technique is that cells can bind to the electrodenon-specifically leading to false fusion events taking place.

A second example of a system for performing cell fusion on individualcells is described in WO01/09297. In this example, cells are manipulatedusing a combination of optical trapping, and pushing the cells withmicro-electrodes. Once the cells are correctly positioned relative toeach other, an electric field is applied to the cells to cause the cellsto fuse.

However, a number of significant drawbacks exist with the apparatus.Firstly, the presence of the laser and associated optics required tomanipulate the cells results in the apparatus being expensive, timeconsuming to configure and complicated to use. Secondly, the electrodesmust be significantly smaller than the cells in size, to allowmanipulation of the cells by pushing. As a result, the electrodes areagain expensive, difficult to construct and extremely fragile, therebyfurther increasing the cost and complexity of the apparatus.

In addition to this, touching the cells with the electrodes can lead toadditional problems, such as burning of the cells. Even in the eventthat a signal is not being applied to the electrodes when the cell ispushed, the electrodes can retain a residual field from when they arelast used. In this case, contact of the cell with the electrode cancause the field to be discharged, thereby damaging the cell.

Finally, the use of the laser trapping and electrodes to manipulatecells is difficult to achieve manually as described in WO01/09297. Thisnot only means that training is required to perform cell fusion usingthe apparatus, but also means the cell fusion process itself can be timeconsuming.

SUMMARY OF THE PRESENT INVENTION

In a first broad form the present invention provides a method of fusingfirst and second cells, the method including:

-   -   a) Selecting the first and second cells;    -   b) Positioning the first and second cells between two electrodes        in a fluid filled fusing container, the first and second cells        being held in suspension separated from each electrode; and,    -   c) Applying a current having a predetermined waveform to the        electrodes to cause the cells to fuse.

Typically the cells are held in suspension between the electrodes.

The method typically includes generating a DEP field, the DEP fieldbeing adapted to urge the cells towards each other.

The predetermined waveform may include a current representing the DEPfield. Alternatively, the method can include applying the DEP to a pairof second electrodes.

The method generally includes:

-   -   a) Applying a DEP current to the pair of second electrodes;    -   b) Positioning the first cell in the fusing container, the        alternating field acting to attract the first cell towards one        of the second pair of electrodes; and,    -   c) Positioning the second cell in the fusing container, the        alternating field acting to attract the second cell towards the        first cell.

At least one of the first and second cells is generally positioned incontact with at least one of the second pair of electrodes.

The method of selecting the first and second cells typically includesusing a pipette to extract:

-   -   a) The first cell from a group of first cells held in a first        container; and,    -   b) The second cell from a group of second cells held in a second        container.

The method of positioning the first and second cells between the twoelectrodes usually includes:

-   -   a) Using the pipette to position the first cell in the fusing        container;    -   b) Using the pipette to position the second cell in the fusing        container, adjacent the first cell;    -   c) Positioning the electrodes such that the first and second        cells are located substantially between the electrodes.

The pipette is typically coupled to:

-   -   a) A drive system adapted to move the pipette with respect to        the first, second and fusing containers; and,    -   b) An actuator adapted to actuate the pipette to thereby expel        or draw in fluid through a port.

In this case, the method usually includes using a controller coupled tothe drive system and the actuator to move and actuate the pipette.

The method of selecting a cell preferably includes causing thecontroller to:

-   -   a) Move the pipette such that the port is adjacent a cell having        predetermined characteristics, the cell being held in fluid        suspension in the respective container;    -   b) Actuate the pipette to draw in fluid through the port,        thereby drawing in the cell and the surrounding fluid.

The method of using the pipette to position the second cell adjacent thefirst cell generally includes causing the controller to:

-   -   a) Move the pipette such that the port is adjacent the first        cell in the fusing container;    -   b) Cause the pipette to expel fluid through the port, thereby        expelling the second into the fluid in the fusing container;    -   c) Move the pipette such that the port is as close as possible        to both the first and second cells;    -   d) Cause the pipette to draw in fluid through the port, thereby        drawing in the first and second cells and the surrounding fluid;    -   e) Cause the pipette to expelling the first and second cells        into the fluid in the fusing container; and,    -   f) Repeat steps (c) to (e) until the first and second cells are        within a predetermined distance.

The electrodes may be coupled to an electrode drive system adapted tomove the electrodes with respect to the fusing containers, in which casethe method typically includes using a controller coupled to theelectrode drive system to position the electrodes in the fusing chamber.

The electrodes may be coupled to a signal generator, in which case themethod of applying the alternating current includes causing the signalgenerator to apply a predetermined waveform to the electrodes.

If the first and second cells having a respective cell type, the methodpreferably includes using a controller coupled to the signal generatorto select the current in accordance with the cell types of the first andsecond cells.

The fist and second cells may be the same type of cell, the first andsecond group of cells being the same group.

In a second broad form the present invention provides apparatus forfusing first and second cells, the apparatus including:

-   -   a) A fluid filled fusing container,    -   b) At least two electrodes adapted to be positioned in the        fusing container in use;    -   c) A selector for:        -   i) Selecting a first cell from a group of first cells held            in a respective container; and,        -   ii) Selecting a second cell from a group of second cells            held in a respective container;        -   iii) Positioning the first and second cells in the fusing            container, the first and second cells being held in            suspension; and,    -   d) A signal generator coupled to the electrodes, the signal        generator being adapted to cause a field having a predetermined        waveform to be generated between the electrodes, thereby causing        the cells to fuse.

The selector is preferably a pipette.

The apparatus generally further includes:

-   -   a) A drive system adapted to move the pipette with respect to        the first, second and fusing containers; and,    -   b) An actuator adapted to cause the pipette to expel or draw in        fluid through a port.

The electrodes may be coupled to the fusing container.

Alternatively the apparatus can include an electrode drive systemadapted to move the electrodes with respect to the fusing containers.

The current waveform typically includes a fusion pulse, the signalgenerator being adapted to apply the fusion pulse to the electrodes togenerate an electric field pulse thereby causing the cells to fuse.

The current waveform preferably also includes a DEP current, the signalgenerator being adapted to apply the DEP current to the electrodes togenerate a DEP field thereby urging the cells towards each other.

The apparatus may include a pair of second electrodes, the pair ofsecond electrodes being coupled to a second signal generator, the secondsignal generator being adapted to generate a DEP current, the DEPcurrent being applied to the pair of second electrodes to generate a DEPfield thereby urging the cells towards each other.

In this case, the pair of second electrodes being provided on the fusingcontainer surface.

The apparatus also typically includes a controller adapted to controlthe fusing of the cells by controlling operation of at least one of:

-   -   a) The pipette;    -   b) The electrodes; and,    -   c) The signal generator.

The controller typically includes a processor coupled to at least oneof:

-   -   a) The drive system and the actuator, the processor being        adapted to move and actuate the pipette;    -   b) The electrode drive system, the processor being adapted to        move the electrodes; and,    -   c) The signal generator, the processor being adapted to cause        the signal generator to generate the field having the        predetermined waveform.

The controller may include a detector adapted to detect the position ofcells within the containers, in which case the processor can beresponsive to the detector to move at least one of the electrodes andthe pipette in response to the position of detected cells.

Alternatively, or additionally, the processing system may include aninput for receiving input commands from a user.

The processor can be coupled to a store for storing waveform datarepresenting a number of different predetermined waveforms, theprocessor being adapted to select one of the number of predeterminedwaveforms in response to the input commands received from the user.

The processor can also being adapted to move at least one of theelectrodes and the pipette in response to the input commands receivedfrom the user.

Typically the controller is adapted to cause the cells to fuse bycausing the apparatus to perform the method of the first broad form ofthe invention.

In a third broad form the present invention provides, a controller forcontrolling apparatus for fusing first and second cells, the apparatusincluding:

-   -   a) A fluid filled fusing container;    -   b) At least two electrodes;    -   c) A selector;    -   d) A signal generator coupled to the electrodes;    -   Wherein, in use, the controller is adapted to cause the cells to        fuse by:        -   i) Causing the selector to:            -   (1) Select a first cell from a group of first cells held                in a respective container; and,            -   (2) Select a second cell from a group of second cells                held in a respective container; and,            -   (3) Position the first and second cells in the fusing                container between the electrodes, the first and second                cells being held in suspension;        -   ii) Positioning the electrodes in the fusing container; and,        -   iii) Causing the signal generator apply a field having a            predetermined waveform to the electrodes, thereby causing            the cells to fuse.

The controller can also be adapted to position the cells in the fusingcontainer.

In this case, the controller typically includes a processor coupled toat least one of:

-   -   a) A drive system adapted to move the pipette with respect to        the first, second and fusing containers;    -   b) An actuator adapted to cause the pipette to expel or draw in        fluid through a port;    -   c) An electrode drive system adapted to move the electrodes with        respect to the fusing containers; and,    -   d) The signal generator.

The current waveform typically includes a fusion pulse, the controllerbeing adapted to cause the signal generator to apply the fusion pulse tothe electrodes to generate an electric field pulse thereby causing thecells to fuse.

The current waveform usually includes a DEP current, the controllerbeing adapted to cause the signal generator to apply the DEP current tothe electrodes to generate a DEP field thereby urging the cells towardseach other.

The apparatus can include a pair of second electrodes, the pair ofsecond electrodes being coupled to a second signal generator, thecontroller being adapted to cause the second signal generator togenerate a DEP current, the DEP current being applied to the pair ofsecond electrodes to generate a DEP field thereby urging the cellstowards each other.

The controller is typically adapted to operate for use with apparatus ofthe second broad form of the invention.

In this case, the controller is preferably adapted to cause theapparatus to perform the method of the first broad form of theinvention.

In a fourth broad form the present invention provides a computer programproduct for controlling apparatus for fusing first and second cells, thecomputer program product including computer executable code which whenexecuted by a suitable processing system causes the processing system tooperate as the controller of the third broad form of the presentinvention.

In a fifth broad form the present invention provides a pipette systemfor manipulating particles, the pipette system including:

-   -   a) A nozzle for containing fluid in use, the nozzle including a        port;    -   b) An actuator coupled to the nozzle, the actuator being adapted        to draw in and/or expel fluid through the port; and,    -   c) An electrode coupled to the nozzle adjacent the port, the        electrode being adapted to cooperate with a second electrode to        allow an electric field to be applied to coupled to one or more        particles positioned adjacent the port.

The electrode is usually formed a conductive tube.

The electrode may be formed from a stainless steel tube having adiameter of approximately 10 mm.

The pipette system can include a drive system adapted to move thepipette system to be with respect to a fluid filled container to therebyallow particles to be positioned in or removed from fluid in thecontainer.

The pipette system can include a signal generator coupled to theelectrode for generating a predetermined electric field between theelectrode and a second electrode positioned in the container.

The pipette system typically includes a controller adapted to controlthe drive system, the actuator and the signal generator to thereby applyan electric field to a particle by:

-   -   a) Positioning the particle in the container adjacent the second        electrode using the pipette;    -   b) Positioning the pipette port adjacent the particle in the        container; and,    -   c) Activating the signal generator.

The controller is typically adapted to fuse cells, by:

-   -   a) Positioning a first cell in the container adjacent the second        electrode using the pipette;    -   b) Positioning a second cell in the container adjacent the first        cell using the pipette;    -   c) Positioning the pipette port adjacent the first and second        cells, such that first and second cells are substantially        between the electrodes; and,    -   d) Activating the signal generator to cause a predetermined        field sequence to be applied to the cells, thereby causing the        cells to fuse.

The pipette system generally further includes:

-   -   a) A radiation source; and,    -   b) A waveguide having a first end coupled to the radiation        source and a second end coupled to the nozzle adjacent the port        to thereby allow radiation from the radiation source to impinge        on particles positioned adjacent to the port in use.

The pipette system can include a detector, the detector being adapted todetect radiation emitted by the particle.

The detector can be coupled to the first end of the waveguide, tothereby detect radiation emitted from the particle.

The radiation is typically a laser, although other sources, such as LEDsmay be used.

The waveguide can be a fibre optic cable, or alternatively can beingformed from the nozzle, the nozzle including a shaped portion to allowthe radiation from the radiation source to enter the nozzle and passalong at least a portion of the nozzle, the radiation being emitted fromthe nozzle through the port.

The pipette system generally includes a controller adapted to perform atleast one of:

-   -   a) Activating the actuator to thereby cause fluid to be drawn in        and/or expelled through the port; and,    -   b) Activating the radiation source, to thereby expose a particle        to radiation.

The drive system can be coupled to a controller, the controller beingadapted to recover particles having predetermined properties from thecontainer by:

-   -   a) Positioning the pipette system such that the port is adjacent        to a particle;    -   b) Activating the radiation source to thereby expose the        particle to radiation;    -   c) Detect any radiation emitted by the particle;    -   d) Determine if the particle has the predetermined properties in        accordance with the detected radiation; and,    -   e) In accordance with a successful comparison, activate the        actuator to thereby draw fluid into the nozzle through the port,        thereby recovering the particle.

The actuator can include:

-   -   a) A fluid reservoir;    -   b) A flexible tube coupling the nozzle to the fluid reservoir;    -   c) An arm positioned so as to partially compress the tube;    -   d) An actuator drive system adapted to move the arm so as to        perform at least one of:        -   i) Further compressing the tube to thereby expel fluid from            the port; and,        -   ii) Decompressing the tube to thereby draw fluid in through            the port.

The actuator drive system generally includes:

-   -   a) A first actuator drive for moving the arm with respect to the        tube; and,    -   b) A second actuator drive formed from an arm end portion, the        arm end portion being in contact with the tube in use, the        second actuator drive being adapted to cause the arm end portion        to expand or contract.

The pipette system usually includes a controller coupled to the actuatordrive system, the controller being adapted to operate the actuator drivesystem to thereby draw fluid in or expel fluid through the port.

The drive system can be coupled to the controller, the controller beingadapted to recover particles from the fluid by:

-   -   a) Positioning the pipette system such that the port is adjacent        to a particle; and,    -   b) Activate the actuator drive system to thereby draw fluid into        the nozzle through the port, thereby recovering the particle.

The tube can be formed from silicon tubing.

BRIEF DESCRIPTION OF THE DRAWINGS

An example of the present invention will now be described with referenceto the accompanying drawings, in which:

FIG. 1 is a block diagram of an example of apparatus for fusing cells;

FIG. 2 is a schematic diagram of the apparatus of FIG. 1;

FIG. 3 is a schematic diagram of the pipette of FIG. 1;

FIG. 4 is a flow chart of an overview of the process of fusing cellsusing the apparatus of FIG. 1;

FIGS. 5A to 5C are a flow chart of the process of fusing cellsimplemented by the apparatus of FIG. 1;

FIGS. 6A and 6B are schematic diagrams of cells being drawn into andexpelled from the pipette of FIG. 3;

FIGS. 6C and 6D are schematic diagrams of the arrangement of theelectrodes and cells in the fusion well during operation of theapparatus of FIG. 1; FIGS. 7A to 7G are examples of pulse sequences thatmay be used in the apparatus of FIG. 1;

FIG. 8A is a schematic plan view of a second example of apparatus forfusing cells;

FIG. 8B is a schematic side view of the modified well array of FIG. 8A;

FIG. 9A is a schematic plan view of a third example of apparatus forfusing cells;

FIG. 9B is a schematic side view of one of the cells shown in FIG. 9A;

FIG. 9C is a schematic perspective view of the first electrodes of FIG.9A;

FIG. 10 is a schematic diagram of the pipette of FIG. 3 modified toinclude an electrode;

FIG. 11 is a block diagram of a modified version of the apparatus ofFIG. 1 adapted to use two of the pipettes shown in FIG. 10;

FIG. 12 is a schematic diagram of the apparatus of FIG. 11;

FIG. 13A is a schematic diagram of the pipette of FIG. 10 modified toinclude a radiation source;

FIG. 13B is a schematic diagram of the pipette of FIG. 3 modified toinclude an alternative radiation source;

FIG. 14 is a schematic diagram of the pipette of FIG. 3 retrieving anumber of cells;

FIG. 15 is a schematic diagram of the pipettes of FIG. 11 positioningcells for subsequent fusion;

FIG. 16 is a schematic diagram of the pipettes of FIG. 11 and fusedcells;

FIG. 17 is a schematic diagram of the pipette of FIG. 3 modified toinclude a radiation source;

FIG. 18A is a schematic diagram of the pipette of FIG. 3 with analternative actuator;

FIG. 18B is a schematic diagram of the operation of the actuator of FIG.18A;

FIG. 18C is a schematic diagram of a first example of the pipette ofFIG. 18A modified for use with a bladder;

FIG. 18D is a schematic diagram of a second example of the pipette ofFIG. 18A modified for use with a bladder;

FIG. 19 is a schematic diagram of a cutting tool used for cutting cells;

FIG. 20 is a block diagram of an example of apparatus for automaticallyfusing cells; and,

FIG. 21 is a schematic diagram of the apparatus of FIG. 20.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An example for apparatus suitable for implementing the present inventionwill now be described with reference to FIGS. 1, 2 and 3.

As shown in FIG. 1, the apparatus includes a processing system 10coupled to an imaging system 11, a control system 12 and a signalgenerator 13. The control system 12 is coupled to a pipette system 14and an electrode system 15, as shown.

The processing system 10 includes a processor 20, a memory 21, aninput/output (I/O) device 22, an image interface 23, a control interface24, and a signal interface 25, coupled together via a bus 26. Theprocessing system may therefore be any one of a number of systems, suchas a suitably programmed computer, specialised hardware, or the like. Inany event, the I/O device typically includes a display, such as acomputer monitor or the like, a keyboard, and one or more other inputdevices such as a mouse, joystick trackball or the like.

The imaging system 11 includes a camera 30 such a CCD camera or the likewhich is coupled to a microscope 31. The imaging system 11 is connectedto the processing system via the image interface 23.

The pipette system 14 includes a pipette shown generally at 33 that iscoupled to the control system 12 via a drive system 32. In use, thecontrol system 12 is coupled to the processor via the control interface24, thereby allowing the drive system 32 to be used to control motionand operation of the pipette, as will be described in more detail below.

Similarly, the electrode system 15 is formed from two electrodes 35coupled to the control system 12 via a drive system 34. Again, thecontrol system 12 allows the drive system 34 to control the position ofthe electrodes, as will be described in more detail below.

In use, the system allows a user to select and move individual cellsusing the pipette system 14. When appropriate cells are placed next toeach other, this allows an electric field to be applied to the cellsusing the electrodes 35 thereby causing the cells to fuse.

In order to achieve this, the apparatus is arranged as shownschematically in FIG. 2 such that the pipette 33 and the electrodes 35may be moved relative to a well array shown generally at 40. This allowscells to be moved between the wells 40, 41, 42, 43, 44,45, 46, 47, 48,as shown.

Movement of the pipette and the electrodes 35 is achieved by operationof the corresponding drive system 32, 34. Accordingly, it will beappreciated that the processing system 10 may be used to controlpositioning of the pipette 33 and the electrodes 35 allowing the pipette33 and the electrodes 35 to be inserted into and positioned within arespective one of the wells 41, . . . , 48.

Furthermore, the microscope 31 is arranged to image selected wells 41, .. . , 48 such that the representation of the contents of a selected wellcan be displayed to the user using the I/O device 22.

In general, the processing system 10 is adapted to control the pipette33 and the electrodes 35 in accordance with input commands received fromthe user via the I/O device 22. In order to achieve this, the processingsystem 10 must be able to perform a number of functions simultaneously,such as:

-   -   Presenting an image of the well array 40 to the user on the I/O        device 22;    -   Responding to commands input via the I/O device 22 to move and,        if required, actuate the pipette system 14;    -   Responding to command inputs via the I/O device 22 to move the        electrodes 35; and,    -   Responding to commands input via the I/O device 22 to apply an        electrical signal to the electrodes 35.

This is achieved by having the processor 20 execute appropriateapplication software which is stored in the memory 21.

The pipette is shown in more detail in FIG. 3. As shown, the pipette 33is formed from a housing 50 defining a chamber that is divided into twoportions 51A, 51B by a piezoelectric element 52, as shown. The chamber51B is coupled by a port 53 to a flexible tube 54. The flexible tube 54includes a male coupling 55 that is adapted to cooperate with a femalecoupling 56 positioned on a shaped glass nozzle 57 having an aperture58, as shown.

In use, the chamber 51B, the port 53, the flexible tube 54 and the glassnozzle 57 are filled with fluid, with the chamber 51A being filled withair and sealed. Applying a current to the piezo-electric element 52, vialeads 59, causes the element to move, with the direction of movementdepending on the polarity of the applied current.

Thus, in use, with the aperture 58 positioned in fluid in one of thewells 41, . . . , 48, causing the piezo-electric element 52 to move inthe direction of the arrow 60 will increase the volume of the chamber51B, thereby causing fluid to be drawn through the aperture 59.Similarly, causing the piezo-electric element 52 to move in thedirection of arrow 61 will decrease the volume of the chamber 51B,thereby causing fluid to be expelled through the aperture 58.

Accordingly, the pipette can be activated to draw in or expel fluidthrough the aperture 58 depending on the polarity of the current appliedto the leads 56. Accordingly, in use, the leads 56 are coupled to eitherthe drive system 32, or a separate activation system, to allow asuitable current to activate the pipette as required.

The manner in which the apparatus is used to fuse cells will now bedescribed.

Overview

An overview of the method of fusing cells in accordance with the presentinvention will now be described with reference to FIG. 4.

In particular, at step 100, the user selects the cells to be fused. Atstep 110, the cells are placed in a fusion well.

At step 120 a predetermined electric field is applied to the selectedcells to cause the cells to fuse.

Cells that are placed in an electric field will distort the field intheir immediate vicinity. The field distortion is dependent on thegeometry and electrical properties of the particle and that of thesurrounding particles. Living cells have interior (cytoplasm) that ishighly conductive, due to the accumulation of ions such as potassium(K+) ions, and a relatively high dielectric constant. The membranesurrounding has a very low conductivity and a lower dielectric constant.

Accordingly, the degree of the distortion of the field both inside andoutside of the cell is a very strong function of the frequency of theapplied electric field. As a result when placed in a non-uniformelectric field cells will experience a force whose magnitude anddirection will vary in a complicated manner with the frequency of theapplied field. This effect can be exploited to selectively manipulateliving cells using radio-frequency alternating electric fields createdvia suitable electrodes. The movement of particles in AC electric fieldsis referred to as ‘dielectropherisis’ (DEP) and is independent of anynet charge on the particle.

The application of the radio-frequency electric fields, typically in theregion 10-10,000 kHz, exerts a positive DEP force on the two cells,urging the cells into close contact with each other. A stronger electricfield is then used in order to induce electrical breakdown of eachcell's membranes at their point of contact. This controlledelectro-poration triggers a process of cell fusion that is somewhat akinto reverse-mitosis. This in turn creates a fused hybrid cell that has agenetic makeup that is a combination of the two original cells that werefused.

The fused cell is then generally placed in a recovery well at step 130before being checked after a predetermined time period to confirm thecell has fused at step 140.

The fused cells can then be collected at 150 and used as required.

DETAILED DESCRIPTION

A detailed example of the method of using the apparatus of the presentinvention will now be described with reference to FIGS. 5A, 5B, 5C and5D.

In this example, the well array 40 includes a target well 41, a partnerwell 42, a washing well 43, a fusion well 44, a recovery well 45 and ahybrid well 46 the purpose of which will be described in more detailbelow.

At step 200 the target and partner cells are placed in respective targetand partner wells. This procedure will generally involve suitablepreparation of the cells, which may be achieved in a number of manners.Thus, for example, this may require that the cells are recovered fromsample plates and washed in appropriate enzyme solutions.

The well array would then be sterilised before appropriate fluids areinserted into the wells to be used. The target and partner cells arethen placed in the target and partner wells, 41, 42 respectively, withthe cells being held in suspension in respective enzyme solutions.

At step 210, the user selects a target cell from the target well 41using the pipette 33. In order to achieve this, the user will arrangethe well array 40 such that the target well 41 is imaged by the imagingsystem. Accordingly, the target well 41 is placed under the microscope31 so that the camera 30 may generate an image signal and transfer thisto the image interface 23. The image signal will then generally undergosome pre-processing in the image interface 23 before being transferredto the processor 20 for any subsequent further processing.

Thus, for example, the image interface 23 may be formed from an imagecapture card, which is used to capture images from incoming imagesignals. The captured image is then formatted by the processor 20 beforebeing presented to the user using the I/O device 22.

The user adjusts the relative position of the microscope 31 and the wellarray until a suitable target cell is shown. The user then uses theprocessing system 10 to control the position of the pipette 33. Inparticular, this is usually achieved by having a joystick I/O device 22,with the processor 20 responding to signals from the joystick togenerate commands which are transferred via the control interface 21 tothe control system 12. The control system will typically be formed froma motion control addressing amplifier, which is coupled to a drivesystem 32, such as suitable stepper or DC servo motors.

By use of appropriate sensitivity control, this allows the position ofthe pipette to be controlled to high degree of accuracy. By arrangingthe microscope such that the pipette is shown in the image presented onthe display, this allows the user to position the pipette 33 with thepipette aperture 58 adjacent the selected cell.

At this point, the user activates the pipette 33 to draw fluid inthrough the aperture 58. The cell and the surrounding fluid will bedrawn into the pipette, allowing the target cell to be removed from thetarget well 41.

Sometimes, it is difficult to separate individual cells within thewells. This can be overcome by repeatedly operating the pipette to causethe pipette to repeatedly draw in and expel fluid via the pipetteaperture 58. Agitation of the fluid medium and repeated movement of thecells through the pipette aperture 58 will usually allow a cell to beseparated from surrounding cells.

An example of this is shown in FIG. 6A, which shows the hydrodynamicstreamlines 70 as fluid is expelled from the pipette aperture 58. Asshown, the hydrodynamic stream-lines, which represent lines of constantforce, spread out away from the pipette aperture 58. Similarly, as thecells, shown at 71, 72, are entrained in the fluid flow, this will tendto cause the cells 71, 72 to separate as they are expelled away from thepipette aperture 58.

In any event, once the user has selected the target cell at step 210,the user washes the target cell in a fusion medium in the washing well43. In order to do this, the pipette containing the respective cell ispositioned in the washing well 43, using the imaging and control system11, 12 to move the pipette 33 as described above. Once the pipette 33 ispositioned inside the washing well 43, the pipette is repeatedlyactivated to cause fluid to be drawn in through and expelled through thepipette aperture 58. In this way, the cell is repeatedly placed in thefusion medium in a washing well 43 and then removed. This action causesthe cell to be washed.

Furthermore, when the user transfers the target and cell to the fusionwell 44 at step 230, this is achieved by positioning the pipette 33 inthe washing well 43 and drawing the target and cell into the pipette 33through the pipette aperture 58. Accordingly, at this point the targetcell is surrounded in fusion medium as opposed to in the mediumcontained in the target well 41.

The user then uses the pipette 33 to place the target cell into thefusion well 44 at step 230. Steps 210 to 230 are repeated for thepartner cell, with the partner cell being placed in the fusion well 44next to the target cell at 230.

As an alternative to performing steps 210 to 230 separately for eachcell, the target and partner cells may be selected from the respectivewells and then washed together in the washing cell 43 being transferredsimultaneously to the fusion well 44.

As will be described in more detail below, it is preferable for thecells 71, 72 to be positioned adjacent to each other. In order toachieve this, it is preferable to first place the target or partner cell71 in the fusion well 44 and then place the other partner or target cell72 adjacent thereto.

In general as adding the second cell 72 will cause fluid to betransferred into the fusion well 44, this also causes movement of thefirst cell 71. It is then generally necessary to repeatedly activate thepipette 33 until the both cells can be drawn in to the pipettesimultaneously. As shown in FIG. 6B, when the cells 71, 72 are drawn into the pipette aperture simultaneously, the hydrodynamic lines of force70 converge as the fluid enters the aperture 58. Accordingly, this drawsthe cells 71, 72 together. The cells can then be expelled from thepipette 33 with the cells being sufficiently close for the fusionprocess to be performed.

In any event, once the user has positioned the target and partner cellsin the fusion well at 230 the user then arranges to place the electrodes35 in the fusion cell 44 at step 240. Again, in order to achieve this,the imaging system 11 is positioned such that the I/O device 22 presentsthe user with an image of the fusion cell 44.

The user can then alter the position of the electrodes 35 by providingappropriate commands via the I/O device 22. Again, this is usuallyachieved by having a respective joystick or the like provide controlsignals to the processor 20. The processor then transfers appropriatecommand signals via the control interface 24 to the control system 12.The control system then activates the drive system 34, thereby casingthe electrodes 35 to move as directed by the user.

An example of the relative positioning of the electrodes 35, the cells71, 72 and the pipette 33 at this stage is shown in FIGS. 6C and 6D,which show a perspective and end on view of the fusion well 44 prior tofusion being performed. Thus, as shown, the cells 71, 72 are positionedclose to each other substantially between the electrodes 35. At thisstage the cells need not be in contact as they will in any event beurged together by the applied electrical field as will be described inmore detail below.

As shown in FIG. 5B, the next step is for the user to determine thesequence of electric fields that are to be applied to the cells at step260 before using the processing system 10 and the signal generator 13 togenerate the determined pulse sequence at step 270.

The manner in which the user determines the electric field will varydepending on the particular implementation of the invention. A firstexample by which this may be achieved is shown in steps 280, 290. Inthis case, the processing system 10 applies a predetermined electricfield to the partner and target cells. The response of the cells in theelectric field is then used to determine the electrical parametersemployed for the DEP electric field (in order to bring the cellstogether). The response can also be used to determine the fusion pulsesequence (including the frequency and amplitude) required to fuse anyparticular pair of cells. In particular, the processing system 10 willapply a field having a predetermined frequency. The frequency can thenbe fine adjusted until an optimum frequency is determined at which theforce that attracts the cells to cells move toward each other is optimalfor the required conditions. This response of the cells to the DEPelectric field will occur due to the generation of electric dipoleswithin the cells, as described above.

The response of the cells to the electric field can be monitored eitherautomatically by having the processor 20 execute appropriate imagerecognition software, or manually by the user. The processor would thenselect a pulse sequence from a number of pulse sequences stored in thememory 32. The pre-programmed pulse sequences would be stored in a lookup table (LUT), or the like, in accordance with the field applied toobtain the desired response. It will be appreciated that thisinformation may need to be determined initially. Accordingly, each timea new lineage of target and partner cell combination is fused, the pulsesequence used to achieve this successfully will be stored in the LUT andthe memory 21, together with information regarding the complete set offusion parameters at which the desired response was observed. Theprocessor 20 can then use the indication of the response to select apulse sequence from the LUT.

Alternatively, the pre-programmed pulse sequences could be stored in theLUT in accordance with each particular type of target and partner cellcombination. Again, this information will need to be determinedinitially. However, by storing the pulse sequence each time a new targetand partner cell combination is fused, this allows the processor 20 toselect a pulse sequence at step 310 in accordance with cell typesprovided by the user at step 300.

In any event, the electric pulse sequences applied to the cells to causethe cells to fuse by DEP at step 320.

At step 330, whilst the cells are still in the fusion well 44 the userexamines the both morphology and the electrical behaviour of the cellsto determine if they have fused to create a fusate cell. If themorphology and behaviour appear favourable to fusion then the fusate istransferred using the pipette 33 to the recovery well 45 at step 360.The initial stages of cell fusion only take a few minutes, typicallyunder ten for most type of cells and accordingly, the user can simplyview the cells on the I/O device 22 and determine from this whether thefusion process has been successful. If it is determined that the cellshave not fused at step 340, the user simply discards the unfused cellswith the pipette 33 at step 350, and returns to select new cells at step210.

Once placed in the recovery well 45 the fusate cell is left forapproximately 45 minutes before again being checked at step 370. Duringthis time, the cell is held in suspension in a suitable culture mediumto encourage cell growth. If it is determined that the fusate cell hasnot completely fused at step 380 then the user discards the unfusedcells using the pipette 33 at step 390, and selects new cells at step210.

Otherwise, the user transfers the fusate cell to a respective hybridwell 46 using the pipette 33 at step 400. The fusate cell is incubatedin the hybrid well at step 410, with the cell being monitored after andduring the incubation process at step 420, to determine if the fusionhas been successful.

Pulse Sequences

As described briefly above, different pulse sequences may be used tocontrol the fusion of the two cells. The generation of different pulsesequences is achieved by having the processor 20 control the signalgenerator 13 in accordance with pre-determined pulse sequences stored inthe memory 21. The pulse sequences are generally stored in data arraysand associated parameters in an LUT, as outlined above or calculatedusing suitable equations and data arrays at the point of fusion. Theprocessor 20 extracts the necessary parameters and the like stored inthe memory 21 and transfers this information to the signal interface 25.

In this example, the signal interface 25 is in the form of an arbitrarysignal generator or the like, which uses the determined parameters todefine a desired pulse sequence. The signal generator thereforegenerates a signal representative of the pulse sequence and transfersthis to a high frequency signal amplifier, allowing the desired pulsesequence to be transferred to the electrodes 35 as required.

It will be appreciated that other forms of pulse sequence generation canalso be used.

In any event, an example of different electrical pulse sequences thatmay be used for fusing cells will now be described. In each of theseexamples, the functions are defined in the temporal domain, t

The basic pulse sequence profiles may be defined in terms of theequations:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=C(t)t ₁ <t<t ₂y ₃(t)=B sin(ωt)>t ₂

-   -   where: A is a constant        -   B is a constant (and may be equal to A)        -   C(t) is the function describing the pulse.

C(t) is typically based on one of the following functions, although itwill be appreciated that this is not essential:C ₁(t)=±KC ₂(t)=Q exp(−αt)C ₃(t)=Q exp(αt)C ₄(t)=Q sin(ξt)

-   -   where: K Q, α and ξ are constants.

Basic pulse sequences can be combined and overlaid to create complexsequences, some examples of which are listed below and are shown inFIGS. 7A to 7G.

FIG. 7A shows a first example of a Basic DC Fusion Pulse Sequenceconsisting of 2 unipolar square pulses, separated by sinusoidal waves.The equations used to govern the generation of these pulse sequences areas follows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=+K t ₁ <t<t ₂y ₃(t)=B sin(ωt)t₂ <t <t ₃y ₄(t)=+K t ₃ <t<t ₄y ₅(t)=A sin(ωt)>t ₄

FIG. 7B shows a second example of a Basic DC Fusion Pulse Sequenceconsisting of a bipolar square pulse, separated by sinusoidal waves. Theequations used to govern the generation of these pulse sequences are asfollows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=+K t ₁ <t<t ₂y ₃(t)=−K t ₂ <t<t ₃y ₄(t)=A sin(ωt)>t ₃

FIG. 7C shows a third example of a Basic AC Fusion pulse consisting of asinusoidal (of differing frequency) of increased amplitude and differingfrequency separated by sinusoidal waves. The equations used to governthe generation of these pulse sequences are as follows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=Q sin(ξt)t ₁ <t<t ₂y ₃(t)=B sin(ωt)t ₂ <t<t ₃y ₄(t)=Q sin(ξt)t ₃ <t<t ₄y ₅(t)=A sin(ωt)>t ₄

FIG. 7D shows a fourth example of a Basic DC and exponential pulseseparated by sinusoidal waves. The equations used to govern thegeneration of these pulse sequences are as follows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=+K t ₁ <t<t ₂y ₃(t)=K+Q exp(−αt)t ₂ <t<t ₃y ₄(t)=A sin(ωt)>t ₃

FIG. 7E shows a fifth example of a Basic DC and exponential pulseseparated by sinusoidal waves. The equations used to govern thegeneration of these pulse sequences are as follows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=+K t ₁ <t<t ₂y ₃(t)=K−Q exp(αt)t ₂ <t<t ₃y ₄(t)=A sin(ωt)>t ₃

FIG. 7F shows a sixth example of a Basic DC pulse sequence convolutedwith a linear curve. The equations used to govern the generation ofthese pulse sequences are as follows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=+K{circle around (x)}(−βt)t ₁ <t<t ₂y ₃(t)=B sin(ωt)t ₂ <t<t ₃y ₄(t)=+K{circle around (x)}(−βt)t ₃ <t<t ₄y ₅(t)=A sin(ωt)>t ₄

Note. An extra DC pulse is shown in FIG. 7F for clarity.

FIG. 7G shows a seventh example of a Basic DC pulse convoluted with anexponential decay curve. The equations used to govern the generation ofthese pulse sequences are as follows:y ₁(t)=A sin(ωt)<t ₁y ₂(t)=+K{circle around (x)}(Q exp(−αt)t ₁ <t<t ₂y ₃(t)=B sin(ωt)t ₂ <t<t ₃y ₄(t)=+K{circle around (x)}(Q exp(−αt))t ₃ <t<t ₄y ₅(t)=A sin(ωt)>t ₄

Note. An extra DC pulse is shown in FIG. 7G for clarity.

SPECIFIC EXAMPLE

An outline of the production of a human-human hybridoma using theapparatus of FIG. 1 will now be described. In general the explanationwill focus on the following staged of the process.

-   -   Preparation of the cells for fusion    -   Setup of the apparatus for fusion    -   Manipulation of the cells in readiness for fusion    -   Electrofusion of the selected pair of cells to obtain hybrid        fusates.        Preparation of the Cells for Fusion.

Peripheral Blood Mononuclear Cells (PBMC) were prepared according to thefollowing protocol: Buffy Coats are obtained from healthy donors(sero-negative for HIV, Hep-B, Hep-C, HTLV-I and Syphilis) from theAustralian Red Cross Blood Bank, Sydney, NSW. PBMC are isolated bydensity centrifugation on Ficoll-Paque™ Plus (Amersham Pharmacia,17-1440-03). The B cells are then isolated for fusion. Untouched B cellsare isolated from PBMC with MACS B Cell Isolation Kit (Miltenyi BioTec,469-01) by magnetic depletion of T cells, NK cells, myeloid cells,basophils, platelets and early erythroid cells. A human myleloma cellline, designated F4 was used as the immortal partner cell.

Set-Up of the Apparatus for Fusion

A 20 ml syringe (#1) was loaded with RPMI media (#2) warmed in anincubator (30 mins at 37 C). Using the syringe a large droplet of thewarmed RPMI solution was deposited into the centre of a Petrie dish(#3). This dish was then placed on the inverted microscope (NikonTE2000) such that it was situated beneath the pipette. The pipettehaving first been sterilized with repeated washings of 70% alcohol/watersolution. The pipette was then lowered so that the tip was immersed inthe droplet of RPMI. One end of a length of silicon tubing(#4) (withsuitable connectors(#5)) was attached to a second syringe and the otherend to the pipette. RPMI was then gently drawn into the pipette andthrough the tubing using the syringe. Care was taken to ensure that noair bubbles formed anywhere along the tubing or in the pipette. Usingthe RPMI filled syringe, fluid was injected into the nozzle of the piezoelectric actuator until it was completely filled and a positive meniscusformed over the nozzle. The second syringe was then gently uncoupledfrom the silicon tubing. Using the first syringe filled with RPMI theuncoupled end of the silicon tubing was topped with fluid until apositive meniscus over the mouth of the connector. The tubing was thencoupled to the piezo electric nozzle 54.

Each pipette nozzle 54 is drawn from capillary tubing (120 μm innerdiameter) from (#7)

The electrodes 35 were then aligned using a graticule until they werespaced ˜400-500 μm apart.

The previously prepared partner cells were then transferred to a singlewell in a 96 well plate (#6) and the lymphocytes were deposited in aseparate well in the same plate.

The pipette was then inserted into the well containing the partner cellsand a suitable partner cell selected. This (single) cell was thentransferred to a fresh well containing RPMI+10% fetal calf serum; FCS.The pipette was then inserted into a well containing the previouslysorted B lymphocytes specific to the target antigen. A suitable Blymphocyte for fusion was then selected. Returning to the previous wellthe lymphocyte cell was expelled from the pipette beside the partnercell. Both cells were then visually inspected for their suitability forfusion.

Manipulation of the Cells Prior to Fusion

The pipette was then used to transfer both cells into a well containingan enzyme solution of 1% pronase plus a sorbitol solution of appropriatepH and osmolarity. The cells were immersed in this medium for fiveminutes before being ‘washed’ in the fusion medium (which is generallyformed from a sorbitol solution of appropriate pH and osmolarity) bygently inhaling and expelling them through the pipette aperture 58 inorder to allow them to acclimatise to the changed environment. Once thecells had adjusted to the change in osmolarity the pipette was then usedto hydrodynamically arrange the cells so that they were within 5-10 μmof each other. The pipette was then removed from the well.

The electrodes 35 were inserted into the well and arranged so that thepreviously arranged cells lay centred and co-linearly between them. Eachelectrode is constructed from a nickel alloy wire of 180 μm diametermanufactured by the Californian Fine Wire Company, California, USA Theconfiguration of the electrodes, their shape and their proximity to thecells are specifically designed so that a suitable electric fieldpattern can be generated in order to induce DEP between the cells.

The electrodes were connected through an amplifier to the arbitrarysignal generator and a series of voltages conforming to differentwaveforms, previously defined by the user, were applied. The firstwaveform applied to the electrodes was sinusoidal and had a frequency of500 kilohertz and an amplitude, post amplifier, of approximately 6V peakto peak. Through phenomena known as dielectropherisis, whereby neutralparticles become polarised in the presence of an alternating,non-uniform, electric field, the cells experienced a force of attractionthat caused them to coalesce.

The amplitude of the field was then increased to 15V peak to peak for aperiod of 5 seconds ensure that good membrane contact was made betweenthe cells. In this increased field there was a slight drift of the cellstowards the upper electrodes, and to counter this the stage of themicroscope was adjusted relative to the electrodes to correct and retainthe cells position between the electrodes.

Electrofusion of the Selected Pair of Cells to Obtain Hybrid Fusates.

Once the cells were suitably arranged a field pattern conforming to thefusion pulse sequence was applied. In this instance the fusion pulsesequence consisted of two pulse trains, each train consisting of 2 DCpulses, of amplitude 90V, (resulting in an electric field ofapproximately 180 kV) each being of 80 μs duration. The pulses wereseparated by a duration of 100 μs, and each train was separated by 500milliseconds, during which in the intervening time a DEP field wasapplied in order to keep the cells in good contact. Post fusion pulsesequence, an increased DEP field was applied in order to maintain goodcontact between the cells whilst the cells fused.

Recovery of the Cells to Growth Medium

The electrodes 35 were the retracted from the fusion well, and thepipette 33 was inserted and manipulated so that the newly created fusedcells were in the vicinity of the pipette aperture 58. The cells werethen inhaled into the pipette and the pipette retracted from the well.In this fashion the cells were transferred to a fresh well containinghybridoma growth media (RPMI+10% FCS). The newly fused cells were theonly cells that were present in this media.

Automation

The above description focuses on manual use of the apparatus, in whichpositioning of the cells, electrodes and pipette are controlled inaccordance with commands input by the user.

However, alternatively the processing system 10 can be adapted tocontrol the apparatus automatically. In order to achieve this, theprocessor 20 executes image recognition applications software stored inthe memory 21. This allows the processing system to use images receivedfrom the imaging system 11 to determine the position of cells within thewells 41, . . . 48, as well as to determine the position of theelectrodes 33 and the pipette 33.

From this, it will be appreciated that the processor 20 and beprogrammed to perform the procedure outlined above automatically.Accordingly, the processing system will be adapted to automaticallyselect target and partner cells in accordance with the appearance of thecell in the image. The cells will then be placed in the fusion well 44to allow the fusion to be performed. Again, during this process theprocessor 20 will control the position of the cells and the electrodes.

The processor then determines the pulse sequence to be applied to thecells, and applies the pulse sequence via the electrodes 35. Once thisis completed the processor 20 can monitor the cells to determine if thefusion process is successful.

It will be appreciated that this may advantageously be achieved usingthe apparatus described in our copending application entitled “CellRecovery”.

Modifications

Experiments have indicated that practically as few as one in seventyfused cells retain the genes needed for mitosis and of these stable celllines a much smaller fraction go onto secrete a protein of interest. Itis therefore desirable to have an apparatus that combined the benefitsof single cell fusion along with high with a throughput of fused cells.Examples of apparatus providing techniques for improving the throughputof the above described apparatus will now be described.

A second example of apparatus suitable for fusing cells will now bedescribed with reference to FIGS. 8A and 8B.

In particular, the apparatus is substantially the same as the apparatusdescribed above with respect to FIGS. 1 to 3. However, in this example,the apparatus includes a modified well array 40 having electrodesincorporated therein. Accordingly, the electrodes 35 are not requiredwith the electrode system 15 utilising the electrodes within the wellarray as will be described in more detail below.

An example of the modified well array is shown in FIG. 8A. As shown, thewell array 80 includes a fusion well 81. Mounted within the fusion well81 are a number of pairs of electrodes 82A, 82B, 83A, 83B, 84A, 84B, 85,85B. Although only four pairs of electrodes have been shown in thisexample, it will be appreciated that a greater number of electrodes maybe used if an appropriately sized fusion well is provided.

The electrodes are typically formed from gold plated to a thickness of˜2 ˜m onto a lower surface 86, as shown in FIG. 8B. The well array mayalso provided with one or more recovery wells 87, 88 as shown.

In use, the predetermined pulse sequences may be applied to the cells71, 72 to be fused using the electrode pairs to 82, 83, 84, 85 as shown.

In use, the user will select the cells 71, 72 to be fused and positionthe cells between a respective pair of electrodes 82 using the pipette,as described above. Once the cells 71, 72 are positioned between theelectrodes 82, the predetermined pulse sequence may be applied to theelectrodes to thereby cause the cells to fuse in the manner describedabove.

From this it will be appreciated that four pairs of cells may bepositioned in the fusion well 81 at any one time, as shown by the dottedlines. Whilst it is possible to fuse the four pairs of cellssimultaneously, it is possible for the field sequence generated eachpair of electrodes 82, 83, 84, 85 to interfere. Accordingly, in somecases it is preferable for each pair of cells to be fused in sequence.

In order to achieve this, the processing system 10 can be adapted toapply a first predetermined pulse sequence to the electrodes 82,followed by a second predetermined pulse sequence to the electrodes 83,etc. It will therefore be appreciated that different field sequences maybe applied to different pairs of electrodes to allow different cells tobe fused within the same recovery well.

A third example of apparatus for fusing cells will now be described withreference to FIGS. 9A, 9B and 9C. In particular, FIG. 9A shows a fusionwell 90 having a first pair of electrodes 91A, 91B and a second pair ofelectrodes 92A, 92B. In use the electrodes 91 are coupled to a firstsignal generator 93 with the electrodes 92A, 92B being coupled to asecond 94. In this case the first and second signal generators replacethe single signal generator 13 shown in FIG. 1, so that the signalgenerators 93, 94 are coupled to the processing system 10, via anappropriate interface 25, to allow their operation to be controlled.

In this example, the electrodes 92A, 92B are used to generate a DEPfield which is adapted to induce a dipole in cells provided at anappropriate location within the fusion well 90. This is used to attractthe cells to a selected one of the electrodes 92A, 92B, thereby allowingthe cells to be positioned accurately within the fusion well.

Accordingly, in use, the AC signal generator 94 will be activated togenerate a DEP field. A pipette is then used to insert cells 95 into thefusion well 90, in a manner similar to that described above. In thiscase, the cell 95 are attracted to the electrode 92A, and will thereforealign as shown. It will be appreciated that this inherent attractionreduces the accuracy with which cells must be positioned within thefusion well 90, compared to in the techniques outlined above, and willoperate to retain the cells 95 in position during subsequent processing.

As shown in FIG. 9B although the cell may contact the electrode 92A, asthe electrode is typically formed from a layer of gold plated onto thebottom of the fusion well 90, the point of contact between the cell 95and the electrode 92A will typically only be very small. Thus, sincethese electrodes are only of the order of a micrometer high, and areonly used to supply the relatively low power DEP field and not thehigher power fusion pulse, as will be described below, the cells willnot be damaged by the procedure, and will be easy to recover from thefusion well 90.

In any event, with a number of first cells 95 positioned in the chamber90 a number of second cells 96 may be positioned adjacent the firstcells 95. In use the dipole induced in the first cells 95 will attractthe second cells 96 to form a number of cell pairs, as shown in FIG. 9A.

Once the required cells are held in position within the fusion well 90,a fusion pulse can be applied to the electrodes 91A, 91B via the firstsignal generator 93. This fusion pulse may consist of a simple DCcurrent applied to the electrodes 91A, 91B, or may be formed from a morecomplex waveform. Similarly, the electrodes 92A, 92B are used togenerate a DEP field in accordance with signals from the second signalgenerator.

Thus, as shown in the signals shown in FIGS. 7A-7G the overall electricfield experienced by the cells consist of a generally alternating DEPfield, with a superimposed fusion pulse formed from a substantially DCfield. In this example, instead of this being achieved using a singleset of electrodes, the fusion pulse is produced by the first set ofelectrodes 91 with the DEP field being produced by the second set ofelectrodes 92.

In this example described, the electrodes 91 can be provided in the cellas fixed electrodes. Alternatively however the electrodes may bepositioned in the cell once the cells 95, 96 are in place. This has anumber of advantages and in particular will avoid stray currents in theelectrodes disturbing the cell placement. An example of the electrodesused in such an arrangement are shown in FIG. 9C.

This arrangement has a number of benefits.

Firstly, allowing the first cells 95 to be placed in a DEP fieldgenerated by the electrodes 92 allows the cells to be arranged far moreeasily in the fusion well 90. In particular, as mentioned above, thecells 95 are held in place by the DEP field, thereby ensuring that theydo not move after placement when further cells are added. This allowsthe cells to be placed as close as five cell diameters apart (althoughthis is not shown in the figure for clarity) allowing a large number ofcells to be aligned accurately in the fusion well 90.

Secondly, the second cells 96 are attracted to the first cells 95 by thegenerated DEP field, thereby causing the cells to naturally align toform cell pairs, as shown at 97. This vastly aids the practical speedwith which cell pairs can be formed at correct locations within thefusion chamber 90. In particular, this allows a number of cell pairs tobe formed in a relatively short space of time such as a couple ofminutes, even using manual operation of the pipette.

Thirdly, as the fusion pulse is provided by the first electrodes 91, thecells will not be damaged by contact with the second electrodes, therebyallowing the cells 96, 96 to be inserted into the fusion well 90 withoutrequiring that they are positioned near to, but out of contact with theelectrodes. As the cells are retained in position well away from thefirst electrodes 91, this allows a higher field strength to be used forthe fusion pulse, which in turn increases the chances of successful cellfusion.

To further enhance this, the DEP field generated by the electrodes 92can be momentarily increased (˜50 ms) as the fusion pulse is generatedbetween the electrodes 91. The purpose of this is to increase thestrength of the dipoles generated in the cells 95, 96, thereby urgingthe cells together with an increased force, to ensure good membrane tomembrane contact between the cells during fusion. This helps increasethe chances of successful cell fusion. Once the fusion pulse is appliedthe increased DEP field can be maintained for a short time after pulsingin order to further aid fusion.

Finally, a further beneficial result of this configuration is that anumber of cell pairs 97 can be arranged in the fusion well 90 andexposed to substantially identical field conditions. This allows a batchof cells to be prepared having substantial identical fusate properties.This helps ensure consistency of the fusate, and allows batches of fusedcells to be produced for experimental purposes.

A fourth example of apparatus for fusing cells will now be describedwith reference to FIGS. 10 to 12.

In this example, apparatus similar to that in FIGS. 1 to 3 is again usedwith one of the electrodes 35 being replaced by an electrode provided onthe pipette 33. An example of the pipette is shown in FIG. 10.

As shown, the pipette is modified by the inclusion of an electrode 100formed from a cylindrical tube 101, and which is coupled to the nozzle57. The electrode 100 is coupled to the nozzle 57 such that the aperture58 is contained in the tube 101 as shown.

In use, the pipette may be used substantially as described above to drawin an expel fluid through the port. This can be used to recover cellsfrom a well allowing the cells to be placed in a fusion well, asdescribed above.

In this example, the fusion well will additionally contain a secondelectrode. The second electrode may be a separate electrode similar toone of the electrodes 35 shown in FIG. 2. The cells can then bepositioned between the electrode 100 and the electrode 35. The signalgenerator is used to apply a predetermined pulse sequence to theelectrodes 100, 35, allowing the cells to be fused as described above.

Alternatively, the electrode may be provided on the underside of thefusion well, in a manner similar to that shown in FIGS. 8A and 8B.

As a further option, a second pipette 33B may be provided with arespective electrode 100B. The resulting apparatus configuration is asshown in FIGS. 11 and 12, with the pipette system 14 being formed fromtwo drive systems 32A, 32B and two pipettes 33A, 33B, as shownAccordingly, in this example, electrodes 100A, 100B provided on thepipettes 33A, 33B, form the electrode system 15.

In any event, an electric field can be generated between the twoelectrodes 100A, 100B to allow cells 71, 72 to be fused in the mannerdescribed above.

It will be appreciated that the provision of a second pipette provides anumber of additional advantages.

In particular, each pipette 33A, 33B is used to position respectivecells 71, 72 adjacent each other by positioning the first cell 71 usingthe first pipette 33A, and then positioning the second cell 72 using thesecond pipette 33B. Once the cells are appropriately positioned, a pulsesequence can be generated between the two electrodes 90A, 90B, therebycausing the cells to fuse.

A number of additional developments can also be implemented for thepipettes. These include the provision of radiation sources such aslasers, LEDs, or the like, and appropriate detectors.

An example of this is shown in FIG. 13A. As shown, the pipette 33includes an LED 102, arranged to direct radiation along the nozzle 57and through the aperture 58, and electrode 100, as shown. The LED istypically coupled to the processing system 10, via leads 103, to allowthe processing system to selectively activate the LED as required. Thisallows a cell 71 adjacent the aperture to be exposed to radiation.

This can be performed for a number of reasons. Thus, for example, thismay be performed to provide simple illumination of the cells. Inparticular, illuminating the cells provides a increased contrast betweenthe cell and surrounding fluid medium, thereby making it easier for thecamera to resolve the cells. This in turn makes images of the cellspresented to the user easier to see, as well as making automateddetection of the cells easier.

In addition to this, the illumination allows cells to be labelled withfluorescent markers or the like, to allow the detectors to detect thecells having predetermined properties as described for example in ourco-pending Patent Application entitled “Cell Recovery”. In this case,visible radiation from an LED may not have sufficient power to cause themarkers to fluoresce. This may be overcome achieved through the use ofan LED operating at ultra-violet wavelengths. Alternatively, this may beachieved using a laser based system as shown in FIG. 13B.

In this example, a laser 105, or other radiation source such as a UVburner with suitable filters, is coupled to an optical fibre 106. Theoptical fibre 106 is coupled to the pipette nozzle 57, using appropriatefixing means, such as a rubber tube (not shown). The optical fibre 106is also coupled to detectors 107, such as photo-diode tubes, viasuitable filters 108.

In use, radiation emitted from the laser is used to expose cells. Anyradiation subsequently reflected from, or emitted by the cells, whichimpinges on the fibre optic cable 106 is transferred to the detectors107. The processing system analyses signals from the detectors and usesthese to select and remove individual cells from a group of cells heldin suspension.

In addition to this, in the example of the system shown in FIG. 16, eachpipette 33A, 33B could be provided with an LED 102A, 102B having adifferent wavelength. This allows the cells to be exposed by differentwavelengths of radiation either to allow cells having differentproperties to be detected, for example through the use of alternativemarkers, or to allow the processing system 10 or the user to determinewhich pipette the respective cell is near.

This also allows the processing system to use the imaging system 11 todetermine from the wavelength of the radiation exposing each cell 71,72, which pipette 33A, 33B is adjacent the cell. This also allows cells71, 72 having different predetermined properties to be detected, byarranging for each cell to respond to a respective wavelength ofradiation, for example by the use of appropriate labels.

This aids in automating the system and provides for a method that allowsa number of cell pairs to be rapidly fused as follows:

-   -   1. Multiple cells in a source well are exposed to radiation from        the LED 102A;    -   2. Cells 71 having predetermined properties are detected by the        processing system 10 and drawn into and stored in the pipette        33A, as shown in FIG. 14.    -   3. Multiple cells in a source well are exposed to radiation from        the LED 102B;    -   4. Cells 72 having predetermined properties are detected by the        processing system 10 and drawn into and stored in the pipette        33B, in a similar fashion.    -   5. Both pipettes 33A, 33B are inserted into a fusion well 44.    -   6. A respective one of each cell type 71, 72, is expelled from        each pipette 33A, 33B at the same time, such that hydrodynamic        forces draw the cells 71, 72 together as shown in FIG. 15.    -   7. The processing system detects the positions of the cells        using the imaging system 11 such that when the cells are        expelled from the respective pipette 33A, 33B the fluid flow is        truncated.    -   8. A DEP field is applied to draw the cells together between the        electrodes, as shown for example in FIG. 12. At this point the        cells are pushed together using an increased (amplitude) DEP        field to aid membrane contact.    -   9. The signal generator 13 applies a predetermined pulse        sequence to the cells 71, 72 via the electrodes 100A, 100B.    -   10. The cells are again pushed together using an increased        (amplitude) DEP field to aid in fusion.    -   11. The pipettes 33A, 33B move to a new position within well.    -   12. Steps 6-11 are repeated as many times as necessary, until a        number of fusates 73 are provided as shown in FIG. 16.    -   13. When all cell pairs have been expelled/fused on of the        pipettes travels back through the well recovering the fusates.    -   14. Fusates are recovered to recovery wells either as single        clones or groups.

It will be appreciated that this technique can be implemented withoutthe presence of the electrodes 100A, 100B, for example by suitablemodification of the pipette shown in FIG. 3.

There also exist techniques for labelling cells that allows them to bemagnetically sorted. In this example, small metal beads are used asmarkers to identify cells of interest. This is achieved by ensuring thatcells having desired properties can be fused to the beads and therebyextracted from a mixture of cells.

This can be achieved for example by coating the beads with an antibodyof interest and then mixing the beads into a culture of cells. Cellsthat are expressing the appropriate receptor on the surface bind to thebeads. The culture is then filtered through a tube, placed in anexternal magnetic field containing thousands of small beads that attractand hold the labelled cells, whilst allowing the unlabelled cells to bewashed through and discarded. Once the external magnetic field isremoved the bound cells can then be washed through the tube and isolatedas desired.

It will be appreciated that this may be achieved on a smaller scaleusing a pipette modified to incorporate an electromagnet.

An example of a suitably modified pipette will now be described withreference to FIG. 17. In this example, the pipette shown generally at110 includes a graphite layer 111 positioned around the pipette nozzle112. A number of cooper windings 113 are provided around a graphite coreto form an electromagnet. In use the copper windings are coupled to a DCsignal generator shown generally at 114, so that the windings act as asolenoid to generate a magnetic field represented by the field lines115.

The copper windings may be provided in a number of layers depending onthe implementation, and may be embedded in a layer of epoxy in order toprevent electrolysis from occurring.

The ends of the wire are connected to a variable DC signal generator anda resistor (R). Passing a current through the wire (taking account ofLenz's Law) will induce a magnetic field, the strength of which isproportional to the applied DC Voltage (V), as given by the equation:$B = {{nuI} = \frac{nuV}{R}}$

-   -   where: n=the number of turns per unit length        -   u=the permeability of free space.

In use, the pipette is positioned near a number of cells which maysuspended in a fluid medium or resting on a substrate 116 as shown at117. In this case, at least some of the cells are attached toappropriate magnetic markers, such as the beads outlined above.

In use, the metal particles, and hence the cells they are attached to,will be attracted into the magnetic field and can therefore be drawninto the pipette in the normal way. This allows cells coupled to themagnetic markers, and hence cells having certain properties to beselected.

It will be appreciated that cells with a higher density of receptors (ahigher number of magnetic markers), should have a larger force exertedon them than cells with less receptors for the same magnetic fieldstrength Therefore as the DC voltage is increased, a larger number ofcells should be drawn into the magnetic fields influence. This fieldgradient can allow for a further sorting criteria.

In order to ensure no wanted cells have been collected, it is possibleto flush out the pipette by expelling fluid from the nozzle. In thiscase, any cells not bound magnetic markers will be expelled from thepipette together with the fluid, whilst the cells bound to markers willbe held in place by the action of the magnetic field. In this case, whenthe selected cells are to be expelled, the magnetic field can bedeactivated, allowing the cells and attached markers to be expelled inthe normal way.

A further development, is for an alternative form of actuator to beused. An example of this form of actuator is shown in FIGS. 18A, 18B.

As shown, in this example, the tube 54 is connected via a stopcock 62and a reservoir 63 to a pump 64. An actuator 65 is positioned adjacentthe flexible tube 54, to allow the tube to be clamped as shown in FIG.18B.

It will be appreciated from this that any form of actuator, such as asolenoid, may be used. However, in this example, the actuator is formedfrom a threaded screw drive 66, coupled a DC or stepper motor 67, whichforms part of the drive system 32. In use, this allows the actuator tobe moved in the direction of the arrow 69, an amount of ±5 mm.

The actuator tip can have a piezo electric stack 68 coupled thereto, toallow fine control (displacement of ±20 μm) of the end of the actuator.Again, the piezo stack forms part of the drive system 32.

In use, the pipette is loaded with a suitable fluid medium by placingthe aperture 58 into a container that has sufficient fluid to fill thesystem. The pump or other such means of drawing fluid through the systemis activated and fluid is drawn through the pipette nozzle 57. When thesystem is loaded and there are no air bubbles present in the tubing, thestopcock 62 is closed to prevent further fluid flow, and the pump 64turned off.

Whilst the aperture 58 is still immersed in the fluid medium, theactuator 65 is adjusted such that the silicon tubing 54 is compressed toabout half its diameter, as shown in FIG. 2B. Thus, in use, with theport 41 positioned in fluid in a well causing the actuator 65 to move inthe direction of the arrow 69 compresses or releases the tubing 54which, in turn, either expels or draws in fluid through the port 41.This allows cells to be recovered from a well as described above withrespect to the pipette of FIG. 3.

Variation on this are shown in FIGS. 18C and 18D. In these examples, theactuator 65 is positioned adjacent a bladder 54A provided in theflexible tube 54. In this case, the bladder has a larger cross sectionalarea than the tube and will therefore contain a greater volume of fluidper unit length compared to the tubing 54. This has two main benefits.In particular, the larger cross sectional area provides for a greaterrange of movement of the actuator. This coupled with the increased fluidvolume in the bladder allows for a greater amount of fluid to bedisplaced when compared to the action of the actuator on the tube 54.

As a result this provides greater control over the amount of fluidexpelled or drawn in through the aperture 58, allowing for greateraccuracy in retrieving individual cells using the pipette.

In this instance, it will be appreciated that by providing sufficientliquid in the bladder, it is not necessary to provide the stopcock 62,the reservoir 63 or the pump 64 as shown in FIG. 18C. In particular, thebladder and pipette can be filled, with an amount of fluid beingexpelled from the bladder before the bladder is positioned so as tocooperate with the actuator, thereby allowing the actuator position tobe adjusted to allow fluid to be drawn in or expelled through theaperture 58.

Alternatively, the bladder can be connected to a stopcock 62, reservoir63 and pump 64, by a tube 54B, as shown in FIG. 18D.

Accordingly, the system described above allows individual cells to beeasily fused. As the cells are manipulated using the pipette as shown inFIG. 3, this makes the cell manipulation far easier than in the priorart. This therefore helps increase the speed and ease with which fusionof individual cells can be performed. Furthermore, the electrodes neednever touch the cells, thereby helping reduce or prevent cell damageprior to or during the fusion process.

In addition to this, the apparatus as a whole is generally lesscomplicated, thereby helping reduce the cost, as well as easing use ofthe apparatus to perform cell fusion. As a result, fusion using thesystem described above can generally be achieved more rapidly andcheaper than in the prior art.

A further development that can be utilised within the examples describedabove is for a cutting tool to be provided to allow cells to be cut, aswell as to allow cells that have adhered to the well surface orelectrodes to be released. An example of a suitable cutting tool isshown in FIG. 19. As shown, the cutting tool includes a support post 120having a blade 121 pivotally mounted thereto by a hinge 122 or otherappropriate connection.

In use, the post is coupled to a micro manipulator (not shown), to allowthe post to be positioned within the respective well. The post can berotated as shown by the arrow 123, allowing the blade to be positionedabove a cell to be cut. If the cell is a free cell 124, the cell willgenerally be held in place using a pipette, or other suitablemanipulator, as shown at 125.

Once positioned, the post is lowered such that the tip of the blade‘bites’ into the soft plastic of the bottom of the plastic plate.Further lowering of the post will cause the blade to pivot around thehinge 122 and ‘guillotine’ through object, such as the cell, placed inits path. Motion is stopped when the blade has cut through the object ofinterest and is completely parallel with bottom of plate.

It will be appreciated that the functionality of the different examplesdescribed above may be combined in any one of a number of arrangements.This allows for example cells to be selected automatically in accordancewith magnetic or radiation sensitive markers. The cells can then bearranged in a fusion well, and fused, with the fusate beingautomatically retrieved and positioned in a recovery well.

A specific example of apparatus for performing automatic cell selectionand fusion will now be described with reference to FIGS. 20 and 21.

As shown in FIG. 20, the control system 12 is further coupled to a stagesystem 16, including a drive system 36 coupled to a stage 37, with theprocessing system 10 being coupled to a stimulation system 17. Thestimulation system 17 is used to stimulate cells, to allow cells havingpredetermined properties to be recovered from a group of cells held insuspension in a selection well.

In order to achieve selection the cells are labelled with markers, whichare adapted to adhere and or permeate only the cells having the requiredpredetermined properties. The stimulation system 17 stimulates themarker cells and thereby identify the cells having the predeterminedproperties. It will be appreciated that the stimulation system 17 may bea radiation based system, similar to that described with respect to FIG.13, or a magnetic based system similar to that described with respect toFIG. 17. The following example will focus on the use of a radiativebased approach.

The arrangement of the apparatus is shown in more detail in FIG. 21.

As shown, the stage 37 includes an aperture 170, having the microscope31 mounted therein. From this it will be appreciated that the microscope31 is typically an inverted microscope.

In use the stage 37 is adapted to receive a selection well 171containing the cells to be recovered. The stage will also receive afusion well 90, positioned over an aperture 172. In use, the selectionwell 171 is positioned on top of the aperture 170, to allow the camera30 to obtain an image of the inside of the selection well 171, via themicroscope 31. In use, the processing system 10 is adapted to controlthe drive system 36, to cause the stage 37 to be move in the directionsshown by the arrows 173, 174.

This allows a representation of the contents of a selected well can becaptured by the processing system 10 using the image interface 23, whichis typically a frame grabber or the like. Images may then be used by theprocessing system to control the drive systems 32, 35 and 36 and thestimulation system 17. Additionally or alternatively, images may bedisplayed to a user using the I/O device 22.

The pipette is positioned adjacent the stage 37 as shown, to allow thenozzle 57 to be inserted into the well 171. The pipette 33 is coupled tothe drive system 32, to allow the pipette to moved with respect to thewell, as shown by the arrows 175, 176, 177. Accordingly, the drivesystem 12 typically includes a micromanipulator system having threeindependently controlled axis with resolution tolerances andrepeatabilities of <5 μm. This system is controlled by dedicatedsoftware executed by the processor 20.

In any event, the cells having the predetermined properties areidentified by exposing the cells to radiation using the radiation source105 coupled to the nozzle 57 via the fibre optic cable 106. This allowsthe detectors 107 to receive radiation emitted by the cells through thefibre optic cable 106 and filters 108, to thereby determine cells havingdesired properties.

It will be appreciated that in the event that the detection of particlesis performed magnetically, this may be achieved as described above withrespect to FIG. 17.

The processing system 10 can then control the pipette system. 14 toremove cells from the selection well 171 and place these in the fusionwell 90, as described above. During this process a DEP field will beapplied to the electrodes 92 to ensure the cells are positioned asrequired. In addition to this, the stage 37 is moved, to allow thecamera 30 to image the fusion well 90 through the aperture 172.

Fusion will then be performed substantially as described above, with thefused cells being removed as required.

Accordingly, the above system describes apparatus suitable formanipulating and fusing cells, and in particular for single cell,mini-bulk or macro-bulk cell fusion. In this regard, the term cells isintended to cover any cells, vectors, particles, molecules, liposomes,and other such vesicles.

In particular, the techniques are particularly advantageous for thepurposes of stem cell fusion, as described for example in our copendingapplication “A Method of Cell Therapy” filed on 30 May 2003.

This allows the techniques to be used for generating tissue or cellsuseful for tissue replacement and/or tissue rejuvenation therapy or arange of organs or tissue areas of the body. The resulting tissue orcells may also secrete or generate a range of cytokines, enzymes,hormones and the like which have improved or more efficacious propertiesrelative to analogous molecules produced from non-fused cells.

In this case, the cells are selected to have desirable properties, suchthat the generated fusate has properties applicable for a specificpurpose.

A suitable list of stem and mature cells and their application for usein transplant and rejuvenation therapy is shown in Table 1. All suchstem and mature cells are contemplated and are encompassed by thepresent invention. As indicated in Table 1, a mature cell may be derivedfrom any human or mammalian or non-mammalian animal or avian speciessuch as from the brain, epidermis, skin, pancreas, kidney, liver, breastlung, muscle, heart, eye, bone, spleen or the immune system. Cells ofthe immune system include CD4+ T-cells, CD8+ T-cells, NK cells,monocytes, macrophages, dendritic cells and B-bells. It should be notedthat the present invention contemplates the fusion of stem cells andmature cells from any source such as a mammal (including human),non-mammalian animal and avian species. Examples of non-human mammalsinclude livestock animals (e.g. sheep, pigs, cows, horses, donkeys,goats), companion animals (e.g. cats, dogs), laboratory test animals(e.g. mice, rats, rabbits, guinea pigs, hamsters) and captured wildanimals. A non-mammalian animal includes a reptile, amphibian, insect,arthropod and arachnids. Avian species include poultry, birds (e.g.ducks, emus, ostriches) and aviary birds. TABLE 1 Cell type ApplicationGeneral Stem cell types Embryonic stem cells Generation of any tissuefor transplant Somatic stem cells Generation of tissue for transplantGerm stem cells Generation of tissue for transplant Human embryonic stemcells Generation of wide variety of tissue for transplant Humanepidermal stem cells Generation of tissue for transplant Tissue-specificcells: Includes both somatic stem cells, mature cells and germ linecells Brain Adult neural stem cells Generation of neural tissue fortransplant Human neurons Generation of neural tissue for transplantHuman astrocytes Generation of neural tissue for transplant EpidermisHuman keratinocyte stem Generation of epidermal type cells tissues suchas hair follicles, sebaceous glands and skin for transplant Humankeratinocyte Generation of epidermal type transient amplifying cellstissues such as hair follicles, sebaceous glands and skin for transplantHuman melanocyte stem cells Generation of epidermal type tissues fortransplant Human melanocytes Generation of epidermal type tissues fortransplant Skin Human foreskin fibroblasts Generation of skin fortransplant Pancreas Human duct cells Generation of insulin-producingcells for transplant Human pancreatic islets Generation ofinsulin-producing cells for transplant Human pancreatic β-cellsGeneration of insulin-producing cells for transplant Kidney Human adultrenal stem Generation of kidney tissue for cells transplant Humanembryonic renal Generation of kidney tissue for epithelial stem cellstransplant Human kidney epithelial Generation of kidney tissue for cellstransplant Liver Human hepatic oval cells Generation ofinsulin-producing cells for transplant Human hepatocytes Generation ofliver tissue for transplant Human bile duct epithelial Generation ofliver tissue for cells transplant Human embryonic endodermal Generationof liver tissue for stem cells transplant Human adult hepatocyte stemGeneration of liver tissue for cells (controversial as to transplantexistence) Breast Human mammary epithelial Generation of mammary(breast) for stem cells transplant Lung Bone marrow-derived stemGeneration of tissue for transplant cells including muscle, cartilage,bone, liver, heart, brain, intestine and lung Human lung fibroblastsGeneration of tissue for transplant including muscle, cartilage, bone,liver, heart, brain, intestine and lung Human bronchial epithelialGeneration of tissue for transplant cells including muscle, cartilage,bone, liver, heart, brain, intestine and lung Human alveolar type IIGeneration of tissue for transplant pneumocytes including muscle,cartilage, bone, liver, heart, brain, intestine and lung Muscle Humanskeletal muscle stem Generation of tissue for transplant cells(satellite cells) Heart Human cardiomyocytes Generation of heart tissuefor transplant Bone marrow mesenchymal Generation of heart tissue forstem cells transplant Simple Squamous Epithelial Generation of heart andvascular cells tissue, for example rebuilding aortic arteries afteraneurysm repairs Descending Aortic Generation of heart and vascularEndothelial cells tissue, for example rebuilding aortic arteries afteraneurysm repairs Aortic Arch Endothelial Generation of heart andvascular cells tissue, for example rebuilding aortic arteries afteraneurysm repairs Aortic Smooth Muscle cells Generation of heart andvascular tissue, for example rebuilding aortic arteries after aneurysmrepairs Eye Limbal stem cells Regeneration of the entire cornealepithelium for transplant Corneal epithelial cells Regeneration of theentire corneal epithelium for transplant Bone Marrow (in some cases besubstituted for cord blood and peripheral blood as a source of some ofthe below stem cells) CD34+ hematopoietic Generation of a wide varietyof stem cells tissues for transplant, including, but not limited to,immune tissue Mesenchymal stem cells Generation of a wide variety oftissues for transplant, including, but not limited to, cardiac tissue,bone, cartilage, muscle, tendon, endothelial tissue, vascular tissue andneural tissue Osteoblasts (precursor is Generation of bone fortransplant mesenchymal stem cell) Peripheral blood Generation of a widevariety of mononuclear progenitor tissues for transplant, includingcells (hematopoietic stem but not limited to cardiac tissue, cells)bone, cartilage, muscle, tendon, endothelial tissue, vascular tissue andneural tissue Osteoclasts (precursor is Generation of bone fortransplant above cell type) Stromal cells Generation of a wide varietyof tissues for transplant, including but not limited to cardiac tissue,bone, cartilage, muscle, tendon, endothelial tissue, vascular tissue andneural tissue Spleen Human splenic precursor Generation of spleen tissuefor stem cells transplant Human splenocytes Generation of spleen tissuefor transplant Immune cells Human CD4+ T-cells Generation of immunecells/tissue for transplant Human CD8+ T-cells Generation of immunecells/tissue for transplant Human NK cells Generation of immunecells/tissue for transplant Human monocytes Generation of immunecells/tissue for transplant Human macrophages Generation of immunecells/tissue for transplant Human dendritic cells Generation of immunecells/tissue for transplant Human B-cells Generation of immunecells/tissue for transplant Nose Goblet cells (mucus Generation ofcells/tissue for secreting cells of the sinus tissue repair nose)Pseudostriated ciliated Generation of cells/tissue for columnar cells(located sinus tissue repair/replacement below olfactory region in thenose) Pseudostratified ciliated Generation of cells/tissue forepithelium (cells that line sinus tissue repair/replacement thenasopharangeal tubes) Trachea Stratified Epithelial cells Generation ofcells/tissue for (cells that line and trachea repair/replacementstructure the trachea) Ciliated Columnar cells Generation ofcells/tissue for (cells that line and trachea repair/replacementstructure the trachea) Goblet cells (cells that Generation ofcells/tissue for line and structure the trachea repair/replacementtrachea) Basal cells (cells that Generation of cells/tissue for line andstructure the trachea repair/replacement trachea) OesophagusCricopharyngeus muscle Generation of cells/tissue for cells oesophagusrepair/replacement Reproduction Female primary follicles Generation ofnatural fertility Male spermatogonium Generation of natural fertility

In terms of using the cells for tissue replacement therapy oraugmentation therapy, at least one population of cells may come from thesubject to be treated or from a histocompatibility matched subject (i.e.an HLA-matched subject). Furthermore, at birth, subjects may store cellsor tissue for the use of the subject (or other suitable subject) laterin life. Such tissue would include placenta tissue, umbilical chordtissue, foreskin, blood or other uteric tissue associated with a fetus.

Additional use is described in the copending application.

Persons skilled in the art will appreciate that numerous variations andmodifications will become apparent. All such variations andmodifications which become apparent to persons skilled in the art,should be considered to fall within the spirit and scope that theinvention broadly appearing before described.

Accordingly, while the above description has focused on cell fusion, itwill be appreciated that the techniques may generally be applied to anycells, vectors, particles, molecules, liposomes, and other suchvesicles.

1) A method of fusing first and second cells, the method including: a) Selecting at least one pair of a first and a second cell; b) Individually positioning each pair of cells such that the respective first and second cells of each cell pair are between two electrodes in a fluid filled fusing container, each cell pair being separated from each electrode and from each other cell pair; and, c) Applying a current having a predetermined waveform to the electrodes to generate a predetermined fusion pulse thereby causing the respective first and second cell of at least one pair of cells to fuse. 2) A method according to claim 1, the cells being held in suspension between the electrodes. 3) A method according to claim 1, the method including generating a DEP field, the DEP field being adapted to urge the cells towards each other. 4) (canceled) 4) A method according to claim 3, the method including applying a current representing the DEP field to a pair of second electrodes. 5) A method according to claim 3, the method including: a) Applying a DEP current to the pair of second electrodes; b) Positioning the first cell in the fusing container, the alternating field acting to attract the first cell towards one of the second pair of electrodes; and, c) Positioning the second cell in the fusing container, the alternating field acting to attract the second cell towards the first cell. 6) A method according to claim 5, at least one of the first and second cells being positioned in contact with at least one of the second pair of electrodes. 7) A method according to claim 1, the method including: a) Selecting the first and second cells using a pipette: b) Using the pipette to position the first cell in the fusing container; c) Using the pipette to position the second cell in the fusing container adjacent the first cell; and, d) Positioning the electrodes such that the first and second cells are located substantially between the electrodes. 9)-11) (canceled) 8) A method according to claim 7, the method of using the pipette to position the second cell adjacent the first cell including causing the controller to: a) Move the pipette such that the port is adjacent the first cell in the fusing container; b) Cause the pipette to expel fluid through the port, thereby expelling the second into the fluid in the fusing container; c) Move the pipette such that the port is as close as possible to both the first and second cells; d) Cause the pipette to draw in fluid through the port, thereby drawing in the first and second cells and the surrounding fluid; e) Cause the pipette to expelling the first and second cells into the fluid in the fusing container; and, f) Repeat steps (c) to (e) until the first and second cells are within a predetermined distance. 9) A method according to claim 1, the electrodes being coupled to an electrode drive system adapted to move the electrodes with respect to the fusing containers, the method including using a controller coupled to the electrode drive system to position the electrodes in the fusing chamber. 10) A method according to claim 1, the electrodes being coupled to a signal generator, the method of applying the current to the electrodes including causing the signal generator to apply a predetermined current to the electrodes. 11) A method according to claim 10, the first and second cells having a respective cell type, the method including using a controller coupled to a signal generator to select the current in accordance with the cell types of the first and second cells. 16)-17) (canceled) 12) Apparatus for fusing first and second cells, the apparatus including: a) A fluid filled fusing container; b) At least two electrodes adapted to be positioned in the fusing container in use; c) A selector for: i) Selecting a first cell from a group of first cells held in a respective container; and, ii) Selecting a second cell from a group of second cells held in a respective container; iii) Individually positioning the respective first and second cells in the fusing container between the electrodes, so that the cell pair is separated from each electrode and each other pair of cells; and, d) A signal generator coupled to the electrodes, the signal generator being adapted to cause a field having a predetermined waveform to be generated between the electrodes, thereby causing the respective first and second cells of at least one pair of cells to fuse. 13) Apparatus according to claim 12, the selector being a pipette. 14) Apparatus according to claim 13, the apparatus further including: a) A drive system adapted to move the pipette with respect to the first, second and fusing containers; and, b) An actuator adapted to cause the pipette to expel or draw in fluid through a port. 15) Apparatus according to claim 12, the electrodes being coupled to the fusing container. 16) Apparatus according to claim 12, the apparatus further including an electrode drive system adapted to move the electrodes with respect to the fusing containers. 17) Apparatus according to claim 12, the current waveform including: a) a fusion pulse, the signal generator being adapted to apply the fusion pulse to the electrodes to generate an electric field pulse thereby causing the cells to fuse, and b) a DEP current, the signal generator being adapted to apply the DEP current to the electrodes to generate a DEP field thereby urging the cells towards each other. 24) (canceled) 18) Apparatus according to claim 12, the apparatus including a pair of second electrodes, the pair of second electrodes being coupled to a second signal generator, the second signal generator being adapted to generate a DEP current, the DEP current being applied to the pair of second electrodes to generate a DEP field thereby urging the cells towards each other. 19) Apparatus according to claim 18, the pair of second electrodes being provided on the fusing container surface. 20) Apparatus according to claim 13, the apparatus further including a controller adapted to control the fusing of the cells by controlling operation of at least one of: a) The pipette; b) The electrodes; and, c) The signal generator. 21) Apparatus according to claim 20, the controller including a processor coupled to at least one of: a) The drive system and the actuator, the processor being adapted to move and actuate the pipette; b) The electrode drive system, the processor being adapted to move the electrodes; and, c) The signal generator, the processor being adapted to cause the signal generator to generate an electrical current having the predetermined waveform. 22) Apparatus according to claim 21, the controller including a detector adapted to detect the position of cells within the containers, the processor being responsive to the detector to move at least one of the electrodes and the pipette in response to the position of detected cells. 30) (canceled) 23) Apparatus according to claim 21, the processor being coupled to a store for storing waveform data representing a number of different predetermined waveforms, the processor being adapted to select one of the number of predetermined waveforms in response to the input commands received from a user. 32)-34) (canceled) 24) A controller for controlling apparatus for fusing first and second cells, the apparatus including: a) A fluid filled fusing container; b) At least two electrodes; c) A selector; d) A signal generator coupled to the electrodes; Wherein, in use, the controller is adapted to cause the cells to fuse by: i) Causing the selector to: (1) Select a first cell from a group of first cells held in a respective container; and, (2) Select a second cell from a group of second cells held in a respective container; and, (3) Individually position the respective first and second cells in the fusing container between the electrodes, the first and second cells being held in suspension, so that the cell pair is being separated from each electrode and each other pair of cells; and, ii) Causing the signal generator apply a field having a predetermined waveform to the electrodes, thereby causing the respective first and second cells of at least one pair of cells to fuse. 25) A controller according to claim 24, the controller being further adapted to position the electrodes in the fusing container. 26) A controller according to claim 25, the controller including processor coupled to at least one of: a) A drive system adapted to move a pipette with respect to the first, second and fusing containers; b) An actuator adapted to cause a pipette to expel or draw in fluid through a port; c) An electrode drive system adapted to move the electrodes with respect to the fusing containers; and, d) The signal generator. 27) A controller according to claim 26, the controller including a detector adapted to detect the position of cells within the containers, the processor being responsive to the detector to move at least one of the electrodes and the pipette in response to the position of detected cells. 39) (canceled) 28) A controller according to claim 26, the processor being coupled to a store for storing waveform data representing a number of different predetermined waveforms, the processor being adapted to select one of the number of predetermined waveforms in response to the input commands received from the user. 29) A controller according to claim 26, the processor being adapted to move at least one of the electrodes and the pipette in response to the input commands received from a user. 30) A controller according to claim 24, the current waveform including at least one of: a) a fusion pulse, the controller being adapted to cause the signal generator to apply the fusion pulse to the electrodes to generate an electric field pulse thereby causing the cells to fuse; and b) a DEP current, the controller being adapted to cause the signal generator to apply the DEP current to the electrodes to generate a DEP field thereby urging the cells towards each other. 31) A controller according to any one of the claim 24, the apparatus including a pair of second electrodes, the pair of second electrodes being coupled to a second signal generator, the controller being adapted to cause the second signal generator to generate a DEP current, the DEP current being applied to the pair of second electrodes to generate a DEP field thereby urging the cells towards each other. 32) A controller according to claim 24, the controller being adapted for use with apparatus including: a) A fluid filled fusing container; b) At least two electrodes adapted to be positioned in the fusing container in use; c) A selector for: i. Selecting a first cell from a group of first cells held in a respective container; and ii. Selecting a second cell from a group of second cells held in a respective container; iii. Individually positioning the respective first and second cells in the fusing container between the electrodes so that the cell pair is separated from each electrode and each other pair of cells; and, d) A signal generator coupled to the electrodes, the signal generator being adapted to cause a field having a predetermined waveform to be generated between the electrodes, thereby causing the respective first and second cells of at least one pair of cells to fuse. 46)-50) (canceled) 33) A pipette system for manipulating particles, the pipette system including: a) A nozzle for containing fluid in use, the nozzle including a port; b) An actuator coupled to the nozzle, the actuator being adapted to draw in and/or expel fluid through the port; and, c) An electrode coupled to the nozzle adjacent to the port, the electrode being adapted to cooperate with a second electrode to allow an electric field to be applied to coupled to one or more particles positioned adjacent to the port. 34) A pipette system according to claim 33, the electrode being formed a conductive tube. 35) A pipette system according to claim 34, the electrode being formed from a stainless steel tube having a diameter of approximately 10 mm. 36) A pipette system according to claim 33, the pipette system including a drive system adapted to move the pipette system to be with respect to a fluid filled container to thereby allow particles to be positioned in or removed from fluid in the container. 37) A pipette system according to claim 36, the pipette system including a signal generator coupled to the electrode for generating a predetermined electric field between the electrode and a second electrode positioned in the container. 38) A pipette system according to claim 37, the pipette system including a controller adapted to control the drive system, the actuator and the signal generator to thereby apply an electric field to a particle by: a) Positioning the particle in the container adjacent the second electrode using the pipette; b) Positioning the pipette port adjacent the particle in the container; and, c) Activating the signal generator. 39) A pipette system according to claim 38, the controller being adapted to fuse cells, by: a) Positioning a first cell in the container adjacent the second electrode using the pipette; b) Positioning a second cell in the container adjacent the first cell using the pipette; c) Positioning the pipette port adjacent the first and second cells, such that first and second cells are substantially between the electrodes; and, d) Activating the signal generator to cause a predetermined field sequence to be applied to the cells, thereby causing the cells to fuse. 40) A pipette system according to claim 39, the pipette system further including: a) A radiation source; and, b) waveguide having a first end coupled to the radiation source and a second end coupled to the nozzle adjacent the port to thereby allow radiation from the radiation source to impinge on particles positioned adjacent to the port in use. 41) A pipette system according to claim 40, the pipette system including a detector, the detector being adapted to detect radiation emitted by the particle. 42) A pipette system according to claim 41, the detector being coupled to the first end of the waveguide, to thereby detect radiation emitted from the particle. 43) A pipette system according to claim 40, the radiation source being a laser. 44) A pipette system according to claim 40, the waveguide being a fibre optic cable. 45) A pipette system according to claim 40, the waveguide being formed from the nozzle, the nozzle including a shaped portion to allow the radiation from the radiation source to enter the nozzle and pass along at least a portion of the nozzle, the radiation being emitted from the nozzle through the port. 46) A pipette system according to claim 40, the pipette system including a controller adapted to perform at least one of: a) activating the actuator to thereby cause fluid to be drawn in and/or expelled through the port; and, b) Activating the radiation source, to thereby expose a particle to radiation. 47) A pipette system according to claim 46, the drive system being coupled to a controller, the controller being adapted to recover particles having predetermined properties from the container by: a) Positioning the pipette system such that the port is adjacent to a particle; b) Activating the radiation source to thereby expose the particle to radiation; c) Detect any radiation emitted by the particle; d) Determine if the particle has the predetermined properties in accordance with the detected radiation; and, e) In accordance with a successful comparison, activate the actuator to thereby draw fluid into the nozzle through the port, thereby recovering the particle. 48) A pipette system according to claim 47, the actuator including: a) A fluid reservoir; b) A flexible tube coupling the nozzle to the fluid reservoir; c) An arm positioned so as to partially compress the tube; d) An actuator drive system adapted to move the arm so as to perform at least one of: i. Further compressing the tube to thereby expel fluid from the port; and, ii. Decompressing the tube to thereby draw fluid in through the port. 49) A pipette system according to claim 48, the actuator drive system including: a) A first actuator drive for moving the arm with respect to the tube and/or a bladder; and, b) A second actuator drive formed from an arm end portion, the arm end portion being in contact with the tube in use, the second actuator drive being adapted to cause the arm end portion to expand or contract. 50) A pipette system according to claim 48, the pipette system including a controller coupled to the actuator drive system, the controller being adapted to operate the actuator drive system to thereby draw fluid in or expel fluid through the port. 51) A pipette system according to claim 50, the drive system being coupled to the controller, the controller being adapted to recover particles from the fluid by: a) Positioning the pipette system such that the port is adjacent to a particle; and, b) Activate the actuator drive system to thereby draw fluid into the nozzle through the port, thereby recovering the particle. 52) A pipette system according to claim 48, the tube being formed from silicon tubing. 71) (canceled) 